Coagulation assays form a group of clinical tests with a wide variety of medical uses. Most coagulation assays are performed on plasma, which is the fluid left after the red blood cells, white blood cells, and platelets (cell fragments from a cell type known as megakarycocytes) are removed either by filtration or centrifugation. The process of clot formation is quite complex, and assays performed on plasma eliminate some of the components involved in clot formation. Historically, the simplification of clotting assays performed on plasma was very important because the technology for measuring clotting had not yet developed to a substantial degree, and the understanding of the coagulation process was very limited. At the present time, however, a more advanced understanding of this clotting process in plasma exists. A large number of automated, highly sophisticated instruments designed specifically for use in plasma-based clotting assays have also been developed over the years. Plasma is also much easier to handle and store than whole blood, and the physical properties of plasma are such that coagulation reactions can be measured with a number of different techniques. A disadvantage of assays performed on plasma is that coagulation is influenced by those components of whole blood which have been removed from the plasma. Plasma-based coagulation clotting assays therefore require the addition of substitutes to attempt to compensate for these missing components.
The addition of substitutes for the missing blood components in a plasma-based assay provides reasonably accurate information regarding in vitro coagulation conditions in whole blood. One frequently performed plasma-based clotting assay is a prothrombin time or PT test. The PT test measures the plasma components of the extrinsic clotting system. The extrinsic clotting system can be thought of as the system which causes blood to clot when a person is cut with a knife, i.e. an open wound. The extrinsic clotting system is triggered by a component known as thromboplastin (or tissue factor), which is located in the linings of the vessels and various cell membranes. In a PT test, thromboplastin is added to the plasma to trigger the formation of a clot. Another frequently performed plasma-based clotting assay is an activated partial thromboplastin time or PTT (or APTT) test. The PTT test is intended to measure the conditions of the intrinsic system. The intrinsic system can be thought of as the clotting system which is triggered when a blood vessel is bruised or damaged. Conducting a PTT test on plasma requires the addition of an activating surface (usually supplied by a negatively charged surface such as glass) and a phospholipid source. The phospholipid substitutes for the activity located on the surface of the platelets known as Platelet Factor 3. Platelet Factor 3 is considerably more effective than phospholipid in promoting clot formation in a PTT test, but Platelet Factor 3 is not used in a PTT test. One frequent complaint with a plasma-based clotting assay is that it does not reliably reflect true in vivo coagulation conditions.
Coagulation assays can also be performed on whole blood, but whole blood coagulation assays are very complex in the information they provide. In whole blood, individual variations exist in hematocrits (the number of red blood cells per unit volume), the number and reactivity of the platelets, and the number and reactivity of white blood cells. In vivo coagulation is affected by each of these components as is in vitro coagulation. The contribution of each of these individual components has, however, not been easily discernible from a clotting assay. In contrast to the more extensive technological developments in plasma-based assays, the technology in whole blood clotting assays has progressed more slowly, primarily because of the historically greater favor for plasma-based assays and because the information available from plasma assays was more managable even though less accurate.
The assignee of the present invention has been instrumental in developing technology useful in performing whole blood coagulation assays. Some of that technology is reflected in U.S. Pat. Nos. 4,000,972, 4,074,971, 4,599,219 and others. The invention of U.S. Pat. No. 4,599,219 can be effectively used to aid in monitoring therapeutic heparin. Generally, therapeutic heparin refers to the use of heparin outside of cardiovascular surgery. Heparin is an agent which extends clotting time of whole blood. In vivo heparin therapy is one very important aspect of modern medicine. In many pathological conditions, accurate knowledge of true intrinsic coagulation conditions can be of considerable aid in assisting with proper treatment in the patient. One feature inherent in U.S. Pat. No. 4,599,219 is the ability to run activated clotting time assays on citrated whole blood samples. Activated clotting time information has been used successfully as an aid in monitoring heparin therapy. In a whole blood activated clotting time test, it is the intrinsic clotting system that is the primary avenue of clot formation. Accordingly, plasma-based PTT tests are not as reliable in supplying information for monitoring heparin therapy as are whole blood activated clotting time tests, which include the effects all of the various components of whole blood which effect clotting.
The standard activated clotting time test on whole blood involves drawing a sample of blood and, as rapidly as possible, placing the sample in a tube containing an activating agent (a negatively charged surface). Generally the activating agent is diatomaceous earth. After mixing the whole blood with the activator agent, the tube is incubated at 37.degree. C. and periodically examined for the formation of a clot. This can be done visually in a manual technique, or the examination can be automated. The apparatus disclosed in U.S. Pat. No. 4,599,299 is an automated system which is capable of running an activated clotting time test on a sample of fresh blood immediately after it has been collected, and which is also capable of performing the activated clotting time test later on a sample of citrated whole blood. Citrated whole blood is whole blood collected in a medium containing a calcium chelating agent, such as sodium citrate (citrate). The whole blood is mixed with the citrate or calcium chelating agent when the sample is collected.
Calcium plays a significant role in the analysis of blood clotting. The act of drawing blood initiates clotting reactions, and unless something is done to stop the process, the clotting times have no diagnostic significance. The formation of a clot is a multi-step process and several of these steps require the presence of calcium ions. By removing the calcium ions, as is the effect when the blood is collected in citrate, the blood can be prevented from clotting. To reinitiate the clotforming process, calcium is added back into the whole blood (recalcification). All plasma-based assays are performed on blood which has been collected into a medium containing a calcium chelating agent. A calcium chelating agent is a chemical which reacts with the calcium in such a fashion that the calcium can no longer function in blood coagulation. The most common chelating agent is citric acid, since it has the fewest side effects on the components of the clotting system. By collecting blood into a medium containing a calcium chelating agent such as citric acid, sample collection and the assay on the citrated sample can be separated by a time period of up to six hours. Plasma obtained from citrated whole blood can also be frozen, for example, for shipment to a laboratory which specializes in exotic clotting assays. Obtaining plasma from citrated whole blood provides the convenience of highly automated instruments, batch processing of large numbers of samples, and significant savings in operator skill level, time utilization and cost. However, the added convenience of plasma-based samples eliminates any consideration in assays of whole blood components which participate in intrinsic coagulation.
Most hospitals and laboratories, if they choose to run whole blood clotting assays, would like to be able to use citrated whole blood. The use of citrated whole blood means that the assay does not have to be run at bedside, and allows the assays to be batch-processed. The apparatus disclosed in U.S. Pat. No. 4,599,299 is readily used to perform activated clotting time tests on recalcified samples of whole blood collected initially in citrate or in some other calcium chelating agent, to thereby provide the convenience for delaying the assay from the time when the blood sample was collected.